1. Cell Biology
  2. Chromosomes and Gene Expression
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Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila

  1. Zaijun Ma
  2. Hui Wang
  3. Yuping Cai
  4. Han Wang
  5. Kongyan Niu
  6. Xiaofen Wu
  7. Huanhuan Ma
  8. Yun Yang
  9. Wenhua Tong
  10. Feng Liu
  11. Zhandong Liu
  12. Yaoyang Zhang
  13. Rui Liu
  14. Zheng-Jiang Zhu  Is a corresponding author
  15. Nan Liu  Is a corresponding author
  1. Shanghai Institute of Organic Chemistry, China
  2. University of Chinese Academy of Sciences, China
  3. State Key Laboratory of Medical Genomics, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, China
  4. Texas Children’s Hospital, United States
  5. Baylor College of Medicine, United States
  6. Singlera Genomics, China
Research Article
Cite this article as: eLife 2018;7:e35368 doi: 10.7554/eLife.35368
9 figures, 1 table and 6 additional files

Figures

Figure 1 with 1 supplement
Adult-onset fidelity loss results in epigenetic drift of H3K27me3.

(A) H3K27me3 peak profiles are comparable between young and aged animals. A scatter plot (top panel) of 331 peaks and a genome browser mini-view (bottom, left panel) illustrated that H3K27me3 had comparable peak profiles between young and aged animals. Peaks were identified from four biological replicates of 3d and 30d male flies using Homer. Data plotted are mean values of peak region at 15d (X-axis) or 30d (Y-axis) as compared to 3d. Each dot on the plot represents a peak locus. For quantitative comparison, the dm6 mapped reads were normalized to a scale factor using deeptools function bamCoverage (see Materials and methods for further details). Majority of peak regions were well maintained during aging (purple). Bar chart (bottom, right panel) showed that the number of Reference-adjusted Reads Per Million (RRPM) using ChIP-Rx datasets, exhibiting a progressive increase of H3K27me3 level with age. ChIP-seq was from muscle tissues of 3-, 15-, and 30d-old male flies. Genotype: 5905. (B) H3K27me3 modification undergoes dramatic gain or loss during development. A scatter plot (top panel) of 300 peaks showed dynamic changes of H3K27me3 signals during embryo, larvae, and pupae. Peaks were identified from two biological replicates of three developmental stages. Data plotted are mean values of peak region at larva (X-axis) or pupa (Y-axis) as compared to embryo. Each dot on the plot represents a peak locus. A scale factor normalized dm6 mapped reads were used to generate the mean value of each peak. Method same as (A). As illustrated by the genome browser mini-view for select genes (bottom panel), H3K27me3 modification was either embryo-specific (black) or selectively decreased in larvae (red), or progressively increased from embryo, larvae to pupae (blue). ChIP-seq was from whole embryo, larvae, and pupae. Genotype as in (A). (C) and (D) H3K27me3 modification increases with age. Scatter plot showed H3K27me3 levels for protein-coding genes (transcriptional start site to transcriptional termination sites as annotated in dm6) of in 3d as compared to 15d (C) and 30d (D). (wilcoxon signed-rank test, p<2.2e-16). The dm6 mapped reads were normalized to a scale factor to compare the relative H3K27me3 level quantitatively. Each dot on the plot represents a single gene locus. X- and Y-axis represented log2 mean value of gene’s reference-adjusted reads. Contour lines indicated that H3K27me3 signals were higher in aged flies compared to 3d-old flies. ChIP-seq and genotype as in (A). (E) Inter-peak genes receive relatively more H3K27me3 modification during aging. We used bootstrapping to generate the mean reference-adjusted ChIP intensity of peak genes or inter-peak genes (10,000 draws with replacement of n = 500). Violin plots represent the bootstrapped mean H3K27me3 level of inter-peak genes (left) and peak genes (right) at 3d, 15d, and 30d. In aging flies, inter-peak genes gained relatively more signal (15d: 36.4%, 30d: 48.4%) than peak genes (15d: 9.4%, 30d: 13.3%). Net gain of H3K27me3 signals during aging was calculated by a subtraction of the mean signal intensity between aged and 3d. Bootstrapped 95% confidence intervals: 3d inter-peak genes: [0.758, 1.017]; 15d inter-peak genes: [1.023, 1.401]; 30d inter-peak genes: [1.149, 1.562]; 3d peak genes: [3.349, 3.739]; 15d peak genes: [3.681, 4.075]; 30d peak genes: [3.817, 4.230]. ChIP-seq and genotype as in (A). (F) H3K27me3 modification during aging has reduced selectivity. Genome browser view of a 5.5 Mb region in the chromosome 3R was shown. H3K27me3 occupancy overlaid between 3d (grey), 15d (cyan), and 30d (pink) (top panel). H3K27me3 modification during aging was shown by deducting 3d signals from those at 15d (middle panel) and by deducting 15d signals from those at 30d of age (bottom panel). The increased modifications had no direct correlation with pre-existing peaks. Black boxes and lines represented peak regions, corresponding to their chromosomal locations. ChIP-seq and genotype as in (A). (G) Violin plots indicate that signals are highly preferential at the peak regions at 3d (left, about 323% more signal compared to inter-peak), but for signals gained during aging, selectivity is dramatically reduced (right, only about 9% more signal acquired by peak than inter-peak). Method same as (E). The proportion of more signals in peak genes was computed by a subtraction of the mean signal intensity between peak genes and inter-peak genes. Bootstrapped 95% confidence intervals: 3d inter-peak genes: [0.758, 1.017]; 3d peak genes: [3.349, 3.739]; late-onset (15d-3d) inter-peak genes: [0.295, 0.315]; (15d-3d) peak genes: [0.320, 0.345]; (30d-3d) inter-peak genes: [0.430, 0.452]; (30d-3d) peak genes: [0.468, 0.493]. ChIP-seq and genotype as in (A).

https://doi.org/10.7554/eLife.35368.002
Figure 1—figure supplement 1
Global occupancy of H3K27me3 modification is preserved with age.

(A) Western blot (top) and quantification (bottom) show an increase of H3K27me3 with age in muscle tissues. (mean ±SD of three biological repeats; student t-test). Genotype: 5905. (B) Quantitative ChIP-Rx protocol. A fixed amount of the mouse epigenome was introduced as the spike-in reference, thereby allowing quantitative and comparative assessment of the fly epigenomes. (C) Venn diagram shows the number of overlapped H3K27me3 enriched peaks and shared bases in peak regions from four replicates. ChIP-seq was from muscle tissues of 3d-old male flies. Genotype as in (A). (D) The distribution of H3K27me3 in genome is preserved in muscle tissues with age. Cistrome correlation tools were used to generate the heatmap according to pair-wise correlation coefficients. Pair-wise Pearson correlations (left panel) of genome-wide H3K27me3 levels were calculated at windows of 1Kbps. Venn diagram (right panel) shows the number of shared regions in H3K27me3 enriched regions between 3d and 30d WT flies. Peak region reproducibly identified by four replicates were used to generate the Venn diagram. ChIP-seq was from muscle tissues of 3d- and 30d-old male flies. Genotype as in (A).

https://doi.org/10.7554/eLife.35368.003
Figure 2 with 1 supplement
PRCs-deficient animals have extended lifespan.

(A) PRCs-deficient animals have extended lifespan. H3K27me3 western blot (left), H3K27me3 quantification (middle), and lifespan curve (right) for PRC single mutants. PRC2 heterozygous mutants of indicated genotype reduce H3K27me3 levels and extend lifespan; PRC1 heterozygous mutants of indicated genotype extend lifespan without changing H3K27me3 levels. To name new mutant, a superscript amended to the gene contained a letter c denoting CRISPR/Cas9 method followed by the size of genomic deletion. All mutants have been backcrossed with WT for five times to ensure a uniform genetic background. See also Figure 2—figure supplement 1A. Western blot was from head tissues of 3d-old male flies. (for H3K27me3 quantification: mean ±SD of three biological repeats; student t-test; for lifespan assay: 25°C; n ≥ 200 per genotype for curve; log-rank test). (B) Pair-wise combination of PRC2 trans-heterozygous double mutants of indicated genotype results in stronger effects in H3K27me3-reduction and life-extension. See also Figure 2—figure supplement 1B. Western blot was from head tissues of 3d-old male flies. (for H3K27me3 quantification: mean ±SD of three biological repeats; student t-test; for lifespan assay: 25°C; n ≥ 200 per genotype for curve; log-rank test).

https://doi.org/10.7554/eLife.35368.004
Figure 2—figure supplement 1
PRCs-deficient animals have extended lifespan.

(A) PRCs-deficient animals have extended lifespan. PRC2 heterozygous mutants of indicated genotype reduce H3K27me3 levels and extend lifespan (top of the dashed line); PRC1 heterozygous mutants of indicated genotype extend lifespan without changing H3K27me3 levels (bottom of the dashed line). See also Figure 2A. Western blot was from head tissues of 3d-old male flies. (for H3K27me3 quantification: mean ±SD of three biological repeats; student t-test, *p<0.05, **p<0.01, ***p<0.001; for lifespan assay: 25°C; n ≥ 200 per genotype for curve; log-rank test, ***p<0.001). (B) Pair-wise combination of PRC2 trans-heterozygous double mutants of indicated genotype results in stronger effects in H3K27me3-reduction and life-extension. See also Figure 2B. Western blot was from head tissues of 3d-old male flies. (for H3K27me3 quantification: mean ±SD of three biological repeats; student t-test, *p<0.05, **p<0.01, ***p<0.001; for lifespan assay: 25°C; n ≥ 200 per genotype for curve; log-rank test, ***p<0.001).

https://doi.org/10.7554/eLife.35368.005
Figure 3 with 1 supplement
Long-lived PRC2 mutants diminish the epigenetic drift of H3K27me3 during aging.

(A) Circos plot of the H3K27me3 epigenome illustrates peak profiles that are highly preserved with age and in PRC2 mutants. Black boxes and lines (innermost circle) represented common peak regions, corresponding to their chromosomal locations. Chromosome ideogram was in grey (outermost ring). PRC2 target genes previously found in cells and during development were shown next to their epigenomic loci. ChIP-seq was from muscle tissues of 3d- and 30d-old male flies. Genotypes: WT: 5905 and Pclc421/+; Su(z)12 c253/+. (B) H3K27me3 modification decreases in PRC2 mutants. Scatter plot showed H3K27me3 levels for gene bodies of all protein-coding genes in Pclc421; Su(z)12c253 as compared to WT. (wilcoxon signed-rank test, p<2.2e-16). Each dot on the plot represents a single gene locus. X- and Y-axis represented log2 mean value of genes’ reference-adjusted reads. Contour lines indicated that H3K27me3 signals were generally higher in WT compared to mutants. ChIP-seq was from muscle tissues of 3d-old male flies. Genotypes as in (A). (C) Inter-peak genes receive relatively less H3K27me3 modification in PRC2 mutants. In PRC2 mutant, inter-peak genes received relatively less signal (25.1%) than those of peak genes (13.7%). Reduction of signals was calculated by a subtraction of the mean signal intensity between WT and PRC2 mutants. Bootstrapped 95% confidence intervals: 3d inter-peak genes: [1.317, 1.861]; 3d Pclc421; Su(z)12c253 inter-peak genes: [0.944, 1.361]; 3d peak genes: [3.946, 4.414]; 3d Pcl421; Su(z)12253 peak genes: [3.364, 3.861]. ChIP-seq and genotypes as in (A). (D) Modification of H3K27me3 during aging has reduced selectivity in PRC2 mutants. Bootstrapped 95% confidence intervals: Pcl421; Su(z)12253 late-onset inter-peak genes: [0.053, 0.147]; Pcl421; Su(z)12253 late-onset peak genes: [−0.778,–0.578]. ChIP-seq was from muscle tissues of 3d and 30d old male flies. Genotypes: Pclc421/+; Su(z)12 c253/+. (E) Age-associated drifting of H3K27me3 is dampened in PRC2 mutants. Scatter plot showed gained H3K27me3 signal for all protein-coding genes in aged Pclc421; Su(z)12c253 and WT flies compared to young flies. Each dot on the plot represents a single gene locus. X- and Y-axis represented signal intensity transformed by Log2. Contour lines indicated that the majority of the gene signals displayed higher levels—thus more rapid changes—in aged WT compared to mutants. ChIP-seq and genotypes as in (A).

https://doi.org/10.7554/eLife.35368.006
Figure 3—figure supplement 1
Long-lived PRC2 mutants diminish the epigenetic drift of H3K27me3 during aging.

(A) Relative quantification of different histone marks between WT and PRC2 mutants. Only H3K27me2/3 were selectively reduced in PRC2 mutants. Western blot was from head tissues of 3d-old male flies. (for H3K27me3 quantification: mean ±SD of three biological repeats; student t-test, *p<0.05, **p<0.01, ***p<0.001). Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12 c253/+. (B) and (C) The modification of H3K27me3 is preserved in muscle tissue between WT and PRC2 mutant. Venn diagram shows the number of shared regions in H3K27me3 enriched regions between WT and PRC2 mutant. Peak region reproducibly identified by two replicates were used to generate the venn diagram. Method as in Figure 1—figure supplement 1D. ChIP-seq was from muscle tissues of 3d-old male flies. Genotypes: WT: 5905. Pclc421/+; Su(z)12 c253/+. escc289/+; E(z)c239/+. (D) H3K27me3 modification decreases in PRC2 mutants. Scatter plot showed H3K27me3 levels for all protein-coding genes in escc289; E(z)c239 as compared to WT. (wilcoxon signed-rank test, p<2.2e-16). Each dot on the plot represents a single gene locus. X- and Y-axis represented log2 mean value of genes’ reference-adjusted reads. Contour lines indicated that H3K27me3 signals were higher in WT compared to mutants. ChIP-seq was from muscle tissues of 3d-old male flies. Genotypes: WT: 5905. escc289/+; E(z)c239/+. (E) Inter-peak genes receive relatively less H3K27me3 modification in PRC2 mutants. In PRC2 mutant, inter-peak genes received relatively less signal (23.8%) than those of peak genes (19.4%). We used bootstrapping to generate the mean reference-adjusted ChIP intensity of peak genes or inter-peak genes (10,000 draws with replacement of n = 500). Bootstrapped 95% confidence intervals: WT inter-peak genes: [2.626, 3.601]; esc289; E(z)239 inter-peak genes: [2.029, 2.627]; WT peak genes: [9.161, 10.367]; esc289; E(z)239 inter-peak genes: [7.363, 8.355]. ChIP-seq and genotypes as in (D). (F) Modification of H3K27me3 during aging has reduced selectivity in PRC2 mutants. Bootstrapped 95% confidence intervals: esc289; E(z)239 late-onset inter-peak genes: [0.551, 0.804]; esc289; E(z)239 late-onset peak genes: [0.532, 0.845]. ChIP-seq was from muscle tissues of 3d and 30d old male flies. Genotypes as in (D). (G) Age-associated drifting of H3K27me3 is dampened in PRC2 mutants. Contour lines indicated that the majority of the gene signals displayed higher levels—thus more rapid changes—in aged WT compared to mutants. ChIP-seq and genotypes as in (C).

https://doi.org/10.7554/eLife.35368.007
Figure 4 with 1 supplement
Epigenetic drifting of H3K27me3 occurs with age in head.

(A) H3K27me3 modification increases with age in head. Scatter plot showed H3K27me3 levels for protein-coding genes (transcriptional start site to transcriptional termination sites as annotated in dm6) of in 3d as compared to 30d. (wilcoxon signed-rank test, p<2.2e-16). The dm6 mapped reads were normalized to a scale factor to quantitatively compare the relative H3K27me3 level. Each dot on the plot represents a single gene locus. X- and Y-axis represented log2 mean value of gene’s reference-adjusted reads. Contour lines indicated that H3K27me3 signals were generally higher in aged compared to 3d-old flies. ChIP-seq was from head tissues of 3d- and 30d-old male flies. Genotype: 5905. (B) Inter-peak genes receive relatively more H3K27me3 modification during aging. We used bootstrapping to generate the mean reference-adjusted ChIP intensity of peak genes or inter-peak genes (10,000 draws with replacement of n = 500). Violin plots represent the bootstrapped mean H3K27me3 level of inter-peak genes (left) and peak genes (right) at 3d and 30d. In aging flies, inter-peak genes gained relatively more signal (30d: 18.8%) than peak genes (30d: 1.1%). Net gain of H3K27me3 signals during aging was calculated by a subtraction of the mean signal intensity between aged and 3d. Bootstrapped 95% confidence intervals: 3d inter-peak genes: [1.907, 2.027]; 30d inter-peak genes: [2.263, 2.402]; 3d peak genes: [6.912, 7.146]; 30d peak genes: [6.985, 7.217]. ChIP-seq and genotype as in (A). (C) Violin plots indicate that signals are highly preferential at the peak regions at 3d (left), but for signals gained during aging, selectivity is dramatically reduced (right). Statistics analysis as in (B). Bootstrapped 95% confidence intervals: 3d inter-peak genes: [1.907, 2.027]; 3d peak genes: [6.912, 7.146]; late-onset (30d-3d) inter-peak genes: [0.2756, 0.505]; (30d-3d) peak genes: [−0.136,0.248]. ChIP-seq and genotype as in (A). (D) H3K27me3 modification decreases in PRC2 mutants. Scatter plot showed H3K27me3 levels for gene bodies of all protein-coding genes in Pclc421; Su(z)12c253 as compared to WT. (wilcoxon signed-rank test, p<2.2e-16). Each dot on the plot represents a single gene locus. X- and Y-axis represented log2 mean value of gene’s reference-adjusted reads. Contour lines indicated that H3K27me3 signals were generally higher in WT compared to mutants. ChIP-seq was from head tissues of 3d-old male flies. Genotypes: WT: 5905 and Pclc421/+; Su(z)12 c253/+. (E) Inter-peak genes receive relatively less H3K27me3 modification in PRC2 mutants. In PRC2 mutant, inter-peak genes received relatively less signal (38.9%) than those of peak genes (18.3%). Statistics analysis as in (B). Bootstrapped 95% confidence intervals: 30d WT inter-peak genes: [0.737, 0.783]; 30d Pclc421; Su(z)12c253 inter-peak genes: [0.4498, 0.4787]; 30d WT peak genes: [2.193, 2.268]; 30d Pcl421; Su(z)12253 peak genes: [1.787, 1.858]. ChIP-seq and genotypes as in (D). (F) Modification of H3K27me3 during aging has reduced selectivity in PRC2 mutants. Statistics analysis as in (B). Bootstrapped 95% confidence intervals: Pcl421; Su(z)12253 late-onset inter-peak genes: [0.0126, 0.026]; Pcl421; Su(z)12253 late-onset peak genes: [−0.076,–0.052]. ChIP-seq was from head tissues of 3d and 30d old male flies. Genotypes: Pclc421/+; Su(z)12 c253/+. (G) Age-associated drifting of H3K27me3 is dampened in PRC2 mutants. Scatter plot showed gained H3K27me3 signal for all protein-coding genes in aged Pclc421; Su(z)12c253 and WT flies compared to young flies. Each dot on the plot represents a single gene locus. X- and Y-axis represented signal intensity. Contour lines indicated that the majority of the gene signals displayed higher levels—thus more rapid changes—in aged WT compared to mutants. ChIP-seq was from head tissues of 3d and 30d-old male flies. Genotypes: WT: 5905 and Pclc421/+; Su(z)12 c253/+.

https://doi.org/10.7554/eLife.35368.008
Figure 4—figure supplement 1
Global occupancy of H3K27me3 modification in head tissues with age and in PRC2-deficiency.

(A) Western blot (top) and quantification (bottom) show an increase of H3K27me3 with age in head tissues. (mean ±SD of three biological repeats; student t-test). Genotype: 5905. (B) The distribution of H3K27me3 in genome is preserved in head tissues with age. Cistrome correlation tools were used to generate the heatmap according to pair-wise correlation coefficients. Pair-wise Pearson correlations (left panel) of genome-wide H3K27me3 levels were calculated at windows of 1Kbps. Venn diagram (right panel) shows the number of shared regions in H3K27me3 enriched regions between 3d and 30d WT flies. Peak region reproducibly identified by two replicates were used to generate the Venn diagram. ChIP-seq was from head tissues of 3d- and 30d-old male flies. Genotypes: WT: 5905. (C) The distribution of H3K27me3 in genome is maintained in head tissue between WT and PRC2 mutant. Statistic analysis as in (B). ChIP-seq was from head tissues of 3d-old male flies. Genotypes: WT: 5905 and Pclc421/+; Su(z)12 c253/+.

https://doi.org/10.7554/eLife.35368.009
Figure 5 with 1 supplement
Transcriptomics links H3K27me3 dynamics to the regulation of glycolytic genes.

(A) Venn diagram shows genes commonly changed in PRC2 single mutants with indicated genotype (left panel). Differential expression genes were computed using DESeq based on normalized count from three biological replicates (p<0.05). Gene ontology (GO) analysis (performed by David) shows glycolysis being the biological processes significantly enriched for genes upregulated in PRC2 long-lived mutants, while oxidation-reduction process is the only pathway enriched for genes down-regulated. The bar graphs represent –log10 (p value) and gene counts in each pathway. RNA-seq was from muscle tissues of 30d-old male flies. (B) WGCNA network analysis reveals a co-regulated change of glycolytic genes. Glycolytic genes were highlighted. WGCNA was used for finding modules of highly correlated genes across mRNA-seq samples. For selected module, Cytoscape was used for visualizing interaction network. Glycolytic genes were highlighted as node, suggesting their expression was changed coordinately. (C) GO analysis shows biological processes related to energy metabolism being significantly decreased with age in WT. Age-dependent mRNA expression change was computed using DESeq based on mRNA-seq data from three biological replicates. GO analysis of 2298 down-regulated genes (cutoff: p<0.05) was performed by David. The top 10 most significantly affected biological processes were shown. The bar graphs represent –log10 (p value) and gene counts in each pathway. RNA-seq was from muscle tissues of 3d- and 30d-old male flies. Genotype: 5905. (D) Genes of glycolysis/gluconeogenesis as annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database show age-associated transcriptional decrease. Y-axis represented normalized read counts transformed by Log2. (41 genes used; see Supplementary file 4; wilcoxon signed rank test, p=0.01). RNA-seq and genotype as in (C). (E) ChIP-qPCR validation. Genomic structures were shown according to the Flybase annotation (www.flybase.org). Along the gene of indicated genotype, a, b, c, and d were underlined, denoting the sites for PCR amplification. Color codes represented ATG (the translation start site), CDS (coding sequence), 5’UTR, and 3’UTR. Different primer sets confirmed that H3K27me3 modification was increased in aged WT and further increased in utx null mutants. H3K27me3 was reduced in PRC2 mutants as compared to WT, with the ratio between mutants and WT smaller than 1 (mean ±SD of three biological repeats; student t-test *p<0.05, **p<0.01; also see Figure 5—figure supplement 1D). ChIP-qPCR was from muscle tissues of 3d- and 30d-old male flies. Genotypes: WT: 5905. PRC2 mutant: Pclc421/+; Su(z)12 c253/+. utxc499/c499. (F–H) qRT-PCR analysis confirms that Tpi and Pgi of glycolytic genes have a decrease with age (F) but become increased in both PRC2 (G) and PRC1 mutants (H). (mean ±SD of three biological repeats; student t-test). qRT-PCR was from muscle tissues of 3d- and 30d-old male flies. Genotypes: WT: 5905. Pclc421/+; Su(z)12 c253/+. Su(z)2 c433/+. (I) H3K27me3 increases in utx deficiency. H3K27me3 western blot (top) and quantification (bottom). (for H3K27me3 quantification: mean ±SD of three biological repeats; student t-test). Western blot was from muscle tissues of 30d-old male flies. Genotypes: WT: 5905. utxc499/c499. (J) utx null animals are short-lived. (for lifespan assay: 25°C; n ≥ 200 per genotype for curve; log-rank test). Genotypes as in (I). (K) qRT-PCR analysis indicates a further decrease of glycolytic genes in utx-deficient animals. (mean ±SD of three biological repeats; student t-test). qRT-PCR was from muscle tissues of 30d-old male flies. Genotypes as in (I).

https://doi.org/10.7554/eLife.35368.010
Figure 5—figure supplement 1
Transcriptomics links H3K27me3 dynamics to the regulation of glycolytic genes.

(A) Number of genes upregulated in PRC2 mutants with indicated genotype. Differential expression genes were computed using DESeq based on normalized count from three biological replicates (cutoff: p<0.05). Note that transcriptional changes were generally mild, and that the majority of changes were genes in the inter-peak regions. RNA-seq was from muscle tissues of 30d-old male flies. (B) Venn diagram shows genes commonly changed in PRC2 single mutants with indicated genotype. Differential expression genes were computed using DESeq based on normalized count from three biological replicates (p<0.05). RNA-seq was from head tissues of 30d-old male flies. (C) Diagram illustrates that the transcriptional changes of genes mediating glucose and the citric acid cycle. Genes and their relative transcriptional changes in 3d- and 30d-old WT and PRC2 long-lived mutants with indicated genotype (cutoff p<0.05; n.s.: not significant). Commonly changed genes, Tpi and Pgi, were highlighted in red. While anaerobic glycolysis yields ATP only, glycolysis at the step of pyruvate formation has a net outcome of both ATP and NADH. (D) ChIP-qPCR of glycolytic genes Tpi (top panel) and Pgi (bottom panel). Genomic structure and gene isoforms were shown according to the Flybase annotation. Along the gene of indicated genotype, a, b, c, and d were underlined, denoting the sites for PCR amplification. Color codes represented ATG (the translation start site), CDS (coding sequence), 5’UTR, and 3’UTR. (mean ±SD of three biological repeats; student t-test). ChIP-qPCR was from muscle tissues of 3d- and 30d-old male flies. Genotypes: WT: 5905. Pclc421/+; Su(z)12 c253/+. utxc499/c499. (E) and (F) Short-lived miR-34 mutants (E) and long-lived piwi mutants (F) demonstrated that changes of the glycolytic genes had no simple correlation with lifespan alterations. (for Differential expression analysis: DESeq, likelihood ratio test, three biological repeats; for lifespan assay: 25°C; n ≥ 200 per genotype for curve; log-rank test). RNA-seq was from head tissues of male flies. Genotypes: WT: 5905. miR-34c120/c120. piwic362/+.

https://doi.org/10.7554/eLife.35368.011
Figure 6 with 2 supplements
Metabolomics shows the effect of PRC2 mutants in reversing glycolytic decline in aging.

(A) Untargeted metabolics identifies that metabolites of the glycolytic pathway (red) are decreased with age. For each metabolite, the median from 10 biological repeats was used to calculate the relative fold change between WT and mutants. Untargeted metabolics was from head tissues of 3d- and 30d-old male flies. Genotype: 5905. (B) Pathway enrichment analysis reveals glycolysis being the biological processes significantly enriched with age. Pathway enrichment analysis was executed using MetaboAnalyst. (Pathway enrichment criteria: wilcoxon rank-sum test, cutoff p<0.01; see Materials and methods for algorithm). Genotype as in (A). (C) Untargeted metabolics identifies that metabolites of the glycolytic pathway (red) are increased in PRC2 mutants. Untargeted metabolics and analysis as in (A). Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12 c253/+. (D) Pathway enrichment analysis reveals glycolysis being the biological processes significantly enriched. (Pathway enrichment criteria: wilcoxon rank-sum test, cutoff p<0.01; see method for algorithm). Genotype as in (C). (E) Lactate is decreased with age (left panel) but increased in PRC2 mutants (right panel). (mean ±SD of 10 biological repeats; wilcoxon rank-sum test). Genotypes: WT: 5905. Pclc421/+; Su(z)12 c253/+. (F) Schematic representation of the glucose flux via glycolysis (left) and citric acid cycle (right). 13C and 12C were indicated for specific metabolites. Dashed rectangles encircled isotopologues pertaining to specific metabolites that contained different number of 13C. (G) Decline of glycolytic metabolites during aging is partially rescued by PRC2 deficiency. For specific metabolite, the median from eight biological repeats was used to calculate the ratio between 8d and 30d of age. (mean ±SD of eight biological repeats; wilcoxon rank-sum test). Metabolomics was from head tissues of 8d- and 30d-old male flies. Genotypes: WT: 5905. Pclc421/+; Su(z)12 c253/+. (H) Age-associated decline of glycolysis is diminished by PRC2 deficiency. 13C-traced glycolytic metabolites were used to determine the level of glycolytic pathway as a whole. In 30d WT, 50.1% of glycolysis remained as relative to its level at 8d of age; in contrast, in PRC2 mutants at 30d of age, 78% of glycolysis remained. (mean ±SD of eight biological repeat; see method for calculation). Metabolomics and genotypes as in (G).

https://doi.org/10.7554/eLife.35368.012
Figure 6—figure supplement 1
Untargeted metabolomics.

(A) Heatmap of metabolites exhibiting differential levels of 3d, 30d WT and 30d PRC2 mutants. Data normalization using support vector regression algorithm. Metabolomics was from head tissues of 3d- and 30d-old male flies. Genotypes: WT: 5905. Pclc421/+; Su(z)12 c253/+. (B) Metabolomic analysis reveals that metabolites of the citric acid cycle show comparable levels between young and aged WT, and between WT and PRC2 mutants. (wilcoxon rank-sum test, n.s.: not significant). Metabolomics and genotypes as in (A).

https://doi.org/10.7554/eLife.35368.013
Figure 6—figure supplement 2
Metabolic glucose flux experiment.

(A) Schematic representation of the glucose flux experiment in adult aging flies. Briefly, flies at 3d (young) and 25d (aged) were switched from 12C-glucose to 13C-glucose food for a fixed 5days, such that the glucose metabolism can be traced by measuring the 13C-labeled metabolites. (B) Relative to the total glucose pool, 13C-glucose accounts for more than 93% and 95% in 8d- and 30d-old animals, respectively, suggesting a near-complete replacement. (mean ±SD of eight biological repeats; wilcoxon rank-sum test, n.s.: not significant). Metabolomics was from head tissues of 8d- and 30d-old male flies. Genotypes: WT: 5905. Pclc421/+; Su(z)12 c253/+. (C) Metabolic flux analysis reveals that the levels of 13C6-labeled glucose are comparable between WT and mutants. (mean ±SD of eight biological repeats; wilcoxon rank-sum test). Metabolomics and genotypes as in (B). (D) and (E) Quantification of isotopologues pertaining to each metabolites of citric acid cycle (D) and their summed intensity (E) indicate no consistent change of the pathway with age and in PRC2-deficiecny. (mean ±SD of eight biological repeats; wilcoxon rank-sum test). Metabolomics and genotypes as in (B).

https://doi.org/10.7554/eLife.35368.014
PRC2 mutants couple enhanced glycolysis with improved adult fitness.

(A) Glucose content shows a slight increase in PRC2 mutants, but this increase is not statistically significant (left panel). Pyruvate, one key end product of glycolysis, is increased (right panel). (mean ±SD of three biological repeats with 10 flies for each measurements; student t-test; n.s.: not significant). Test was from muscle tissues of 30d-old male flies. Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12 c253/+. (B) The ratio of NADPH/NADP+, indicator of the PPP, foliate metabolism, and malic enzyme is decreased in mutants. Test, statistics, and genotype as in (A). (C) ATP and cellular redox levels are decreased with age. (mean ±SD of three biological repeats with 10 flies for each measurements; student t-test). Test was from muscle tissues of 3d- and 30d-old male flies. Genotype: 5905. (D) ATP and cellular redox levels are increased in PRC2 deficient animals. Test, statistics, and genotype as in (A). (E) and (F) Analysis of adult phenotypes reveals that PRC2 mutants have a healthy lifespan. Climbing assay exhibited that, whereas WT and mutants behaved similarly at 3d, with age, mutants had better climbing, reflective of improved mobility (E). Mutants had enhanced resistance to oxidation (F). (for climbing assay: mean ±SD of 10 biological repeats with 10 flies for each repeats; student t-test; for oxidation tests: 25°C; n = 100 per genotype for curve; log-rank test). Genotypes as in (A).

https://doi.org/10.7554/eLife.35368.015
Figure 8 with 1 supplement
Perturbing glycolysis diminishes longevity benefits in PRC2 mutants.

(A) Tpi deficiency significantly diminishes the longevity phenotype of PRC2 trans-heterozygous double mutants. (for lifespan assay: 25°C; n > 200 per genotype; log-rank test). Genotypes: Pclc421/+; Su(z)12 c253/+. Pclc421/+; Su(z)12c253, Tpic511/+. (B) and (C) Oxidation stress test (B) and climbing (C) show that Tpi deficiency partially diminishes the lifespan-benefits of PRC2 mutants. (for oxidation assay: 25°C; n = 100 per genotype for curve; log-rank test; for climbing assay: mean ±SD of 10 biological repeats with 10 flies for each repeat; student t-test). Genotypes as in (A). (D) Tpi deficiency reduces glycolysis of PRC2 mutants. Analysis of specific metabolites revealed a decrease of ATP (left panel), ratio of NADH/NAD+ (middle panel), and ratio of GSH/(GSH +GSSG) (right panel). (mean ±SD of three biological repeats; student t-test). Metabolite analysis was from muscle tissues of 30d-old male flies. Genotypes as in (A). (E–H) Pgi deficiency diminishes the aging benefits mediated by PRC2 mutants, including lifespan (E), oxidative stress (F), locomotion (G), and glycolysis (H). Genotypes: Pclc421/+; Su(z)12 c253/+. Pclc421, Pgic392/+; Su(z)12 c253/+.

https://doi.org/10.7554/eLife.35368.016
Figure 8—figure supplement 1
Tpi- and Pgi-deficient flies have normal adult phenotypes.

(A)-(D) and (F)-(I) Assessments of adult lifespan (A) and (F), oxidation test (B) and (G), climbing ability (C) and (H), and the levels of ATP and the ratio of NADH/NAD+ (D) and (I), show no difference between WT and flies with either Tpi or Pgi heterozygous mutation. (for lifespan assay: 25°C; n > 190 per genotype for curve; log-rank test; for oxidation tests: 25°C; n = 100 per genotype for curve; log-rank test; for climbing assay: mean ±SD of 10 biological for each repeats; student t-test; for analysis of metabolites: mean ±SD of three biological for each repeats; student t-test; n.s.: not significant). Genotypes: WT: 5905. Tpic511/+. Pgic392/+. (E) and (J) Tpi and Pgi mRNA level in triple mutants. (mean ±SD of three biological repeats; student t-test). qRT-PCR was from muscle tissues of 3d-old male flies. Genotypes: Pclc421/+; Su(z)12 c253/+. Pclc421/+; Su(z)12c253, Tpic511/+. Pclc421,Pgic392/+; Su(z)12 c253/+, Tpic511/+.

https://doi.org/10.7554/eLife.35368.017
Figure 9 with 1 supplement
Transgenic increase of glycolytic genes suffices to elevate glycolysis and healthy lifespan.

(A) Tpi protein western blot (top) and quantification (bottom) shows a decrease with age. (mean ±SD of three biological repeats; student t-test). Western blot was from 3d- and 30d-old male flies. Genotype: Tpi (+)/+. (B) Pgi protein western blot (top) and quantification (bottom) shows a decrease with age. Statistics and Western blot as in (A). Genotype: Pgi (+)/+. (C) Tpi protein western blot (top) and quantification (bottom) shows an increase in PRC2 mutants. Statistics and western blot as in (A). Genotype: Tpi (+)/+. Pclc421, Tpi (+)/+; Su(z)12 c253/+. (D) Pgi protein western blot (top) and quantification (bottom) shows an increase in PRC2 mutants. Statistics and western blot as in (A). Genotype: Pgi (+)/+. Pclc421/+; Pgi (+), Su(z)12 c253/+. (E) Transgenic increase of glycolytic genes stimulates glycolysis, as shown by elevated pyruvate, ATP, and NADH/NAD+ ratio compared to age-matched WT (mean ±SD of three biological repeats; student t-test). Metabolite analysis was from muscle tissues of 30d-old male flies. Genotypes: WT: 5905. Tpi (+)/+. Pgi (+)/+. Tpi (+)/+; Pgi (+)/+. (F–H) Transgenic increase of glycolytic genes promotes adult fitness, including lifespan (F), locomotion (G), and resistance to oxidative stress (H). (for lifespan assay: 25°C; n ≥ 200 per genotype for curve; log-rank test; for climbing assay: mean ±SD of 10 biological repeats with 10 flies for each repeat; student t-test; for oxidation assay: 25°C; n = 100 per genotype for curve; log-rank test). Genotypes as in (E). (I) Model. Adult-onset fidelity loss results in epigenetic drift of H3K27me3. Over a chronic timescale, the drifting of H3K27me3 induces transcriptional and metabolic decline including a reduction of glycolysis. Effects of PRC2-deficiency in life-extension can be at least in part attributed to the effect in stimulation of glycolysis, thereby maintaining metabolic health and longevity. Adult lifespan is inherently modulated by the alterations in the levels of H3K27me3, as shown by the corresponding change during natural aging, in utx mutants as well as in PRCs-deficiency.

https://doi.org/10.7554/eLife.35368.018
Figure 9—figure supplement 1
Genomic transgenes for Tpi and Pgi.

(A) and (B) Genomic structure and gene isoforms were shown according to the Flybase annotation. Color codes represented ATG (the translation start site), HA tag, CDS (coding sequence), 5’UTR, and 3’UTR. Positive control was based on the UAS/Gal4 system and overexpression of cDNAs for Tpi and Pgi, respectively. Transient transfection of Tpi and Pgi genomic constructs yielded protein of appropriate size. Western blot was using Drosophila S2R+ cells.

https://doi.org/10.7554/eLife.35368.019

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Cell line
(D. melanogaster)
S2R+This paperFLYB:FBtc0000150
AntibodyH3K27me3
(rabbit polyclonal)
MilliporeCat#07–449;
RRID:AB_310624
AntibodyH3K27me2
(rabbit polyclonal)
AbcamCat#ab24684;
RRID:AB_448222
AntibodyH3K27me
(rabbit polyclonal)
MilliporeCat#07–448
AntibodyH3K27ac
(rabbit polyclonal)
AbcamCat#ab4729;
RRID:AB_2118291
AntibodyH3K9me3
(rabbit polyclonal)
AbcamCat#ab8898;
RRID:AB_306848
AntibodyH3K9ac
(rabbit polyclonal)
MilliporeCat#06–942;
RRID:AB_310308
AntibodyH3K23ac
(rabbit polyclonal)
MilliporeCat#07–355;
RRID:AB_310546
AntibodyH3K4me3
(rabbit polyclonal)
MilliporeCat#07–473;
RRID:AB_1977252
AntibodyH3K4me2
(rabbit polyclonal)
MilliporeCat#07–030;
RRID:AB_310342
AntibodyH3K4me
(rabbit polyclonal)
MilliporeCat#07–436;
RRID:AB_310614
AntibodyH3K36ac
(rabbit polyclonal)
MilliporeCat#07–540
AntibodyH3K36me3
(rabbit polyclonal)
AbcamCat#ab9050;
RRID:AB_306966
AntibodyH3K14ac
(rabbit polyclonal)
MilliporeCat#07–353;
RRID:AB_310545
AntibodyH3K18ac
(rabbit polyclonal)
AbcamCat#ab1191;
RRID:AB_298692
AntibodyH4K20me3
(rabbit polyclonal)
AbcamCat#ab9053
AntibodyH3 (goat polyclonal)AbcamCat#Ab12079
AntibodyHA (rabbit monoclonal)Cell signaling technologyCat#3724
AntibodyTubulin
(rabbit polyclonal)
Medical and biological
laboratories co
Cat#PM054
AntibodyAnti-Rabbit IgGSigmaCat#A9169
AntibodyAnti-Mouse IgGSigmaCat#A4416
AntibodyAnti-Goat IgGAbcamCat#ab6471
Recombinant DNA
reagent
pBID UAS vector: HA-TpiAddgene35198
Recombinant DNA
reagent
pBID UAS vector:
HA-Pgi
Addgene35198
Recombinant DNA
reagent
pBID vector:
HA-Tpi genomic
Addgene35190
Recombinant DNA
reagent
pBID vector:
HA-Pgi genomic
Addgene35190
Recombinant DNA
reagent
pU6b-sgRNARen et al., 2013N/A
Commercial assay or kitTURBO DNA-free kitThermoFisherCat#AM1907
Commercial assay or kitSuperSignal West Pico
Chemilluminescent
Subtrate
ThermoFisherCat#34078
Commercial assay or kitNuclear-Cytosol
Extraction Kit
AogmaCat#9988
Commercial assay or kitSYBR Select Master MixThermoFisherCat#26161
Commercial assay or kitSuperScript III First-strand
synthesis system for RT-PCR
ThermoFisherCat#18080–051
Commercial assay or kitChIP-Grade Protein A/G
Plus Agarose
ThermoFisherCat#26161
Commercial assay or kitQIAquick PCR Purification KitQIAGENCat#28106
Commercial assay or kitQubit dsDNA HS Assay KitThermoFisherCat#Q32851
Commercial assay or kitHigh Sentivity DNA Analysis KitsAgilent TechnologiesCat#5067–4626
Commercial assay or kitNEBNext DNA Library Prep KitNew England BiolabsCat#E7370L
Commercial assay or kitNEBNext Poly(A) mRNA
Magnetic Isolation Module
New England BiolabsCat#E7490L
Commercial assay or kitNEBNext Multiplex Oligos
for Illumina
New England BiolabsCat#E7600S
Commercial assay or kitNEBNext Ultra RNA library
Prep Kit for Illumina
New England BiolabsCat#E7530L
Commercial assay or kitRNase A/T1 MixThermoFisherCat#EN0551
Commercial assay or kitGlucose (HK) Assay KitSigmaCat#GAHK20
Commercial assay or kitNAD/NADH Quantitation KitSigmaCat#MAK037
Commercial assay or kitNADP/NADPH
Quantitation Kit
SigmaCat#MAK038
Commercial assay or kitENLITEN ATP Assay SystemPromegaCat#FF2000
Commercial assay or kitPyruvate Assay KitSigmaCat#: MAK071
Commercial assay or kitGSH/GSSG Ratio
Detection Assay
AbcamCat#ab13881
Commercial assay or kitAmicon Ultra 0.5 mL
centrifugal filters
SigmaCat#Z677108-96EA
Chemical compound,
drug
Tris-buffer (1 mol/L, pH8.5)Sangon BiotechCat#SD8141
Chemical compound,
drug
TRIZol RreagentThermoFisherCat#15596018
Chemical compound,
drug
ChloroformSinopharm Chemical ReagentCat#10006818
 Chemical compound,
drug
IsopropanolSinopharm Chemical ReagentCat#10006818
Chemical compound,
drug
DEPC-treated waterInvitrogenCat#46–2224
Chemical compound,
drug
NuPAGE 12% Bis-Tris GelThermoFisherCat#NP0342BOX
Chemical compound,
drug
PageRuler Prestained
Protein Ladder
ThermoFisherCat#26616
Chemical compound,
drug
NuPAGE MOPS SDS
Running Buffer
ThermoFisherCat#NP0001
Chemical compound,
drug
Immobilon Transfer
Membranes
MilliporeCat#: IPVH00010
Chemical compound,
drug
GlycinebeyotimeCat#ST085-1000G
Chemical compound,
drug
20 X PBSSangon BiotechCat#B548117-0500
Chemical compound,
drug
37% formaldehyde solutionSigmaCat#F1635
Chemical compound,
drug
Protease inhibitor
cocktail tablets
RocheCat#11697498001
Chemical compound,
drug
RIPA bufferSigmaCat#R-278–500 ML
Chemical compound,
drug
5M NaClThermoFisherCat#AM9760G
Chemical compound,
drug
0.5M EDTAThermoFisherCat#15575–038
Chemical compound,
drug
1M Tris-HClThermoFisherCat#15568–025
Chemical compound,
drug
Triton X-100SigmaCat#T8787-50ML
Chemical compound,
drug
10% SDSSangon BiotechCat#SD8118
Chemical compound,
drug
RNase A/T1 MixThermoFisherCat#EN0551
Chemical compound,
drug
Sodium dicarbonateSigmaCat#S5761
Chemical compound,
drug
Proteinase KSangon BiotechCat#A100706
Chemical compound,
drug
3M Sodium Acetate pH5.5ThermoFisherCat#AM9740
Chemical compound,
drug
Methyl viologen dichloride
hydrate
SigmaCat#856177
Chemical compound,
drug
Hydrogen peroxide 30%Sinopharm Chemical ReagentCat#10011208
Chemical compound,
drug
D(+)-ArabinoseSangon BiotechCat#A600071-0100
Chemical compound,
drug
D-glucoseSangon BiotechCat#A100188-0500
Chemical compound,
drug
Sodium hydroxide
solution
SigmaCat#72068
Chemical compound,
drug
MethanolHoneywellCat#LC230-2.5HC
Chemical compound,
drug
AcetonitrileMerckCat#1.00029.2500
Chemical compound,
drug
WaterHoneywellCat#LC365-2.5HC
Chemical compound,
drug
Ammonium acetateSigma-AldrichCat#73594–25 G-F
Chemical compound,
drug
Ammonium hydroxideSigma-AldrichCat#44273–100 mL-F
Chemical compound,
drug
Sequencing Grade
Modified Trypsin
PromegaCat#V5111
Chemical compound,
drug
Centrifugal Filters
Ultracel 30K
AmiconCat#UFC30SV00
Chemical compound,
drug
Water, LC/MSJ.T.BakerCat#9831–03
Chemical compound,
drug
Formic acid, eluent
additive for LC-MS
SigmaCat#56302–50 ml-f
Chemical compound,
drug
Acetonitrile,LC/MS,4LJ.T.BakerCat#9829–03
Chemical compound,
drug
Ammonium bicarbonateSigmaCat#09830–1 KG
Chemical compound,
drug
UreaSigmaCat#U5128-500G
Chemical compound,
drug
Trizma hydrochlorideSigmaCat#T5941-100G
Chemical compound,
drug
Ammonium formateFluckaCat#17843–50G
Chemical compound,
drug
Lys(6) SILAC yeastSilantesCat#234CXX-SYK6-509-01
Chemical compound,
drug
D-Glucose (U-13C6,99%)Cambridge Isotope Laboratories, Inc.Cat#110187-42-3
Software, algorithmBowtie2Langmead and Salzberg (2012)http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Software, algorithmSamtoolsLi et al. (2009)http://samtools.sourceforge.net/; RRID:SCR_002105
Software, algorithmdeepToolsRamírez et al., 2014https://deeptools.github.io/
Software, algorithmhomerHeinz et al., 2010http://homer.ucsd.edu/homer/ngs/index.html; RRID:SCR_010881
Software, algorithmbwtoolPohl and Beato, 2014https://users.dcc.uchile.cl/~peortega/bwtool/; RRID:SCR_003035
Software, algorithmGalaxyGrüning et al., 2017https://github.com/bgruening/galaxytools; RRID:SCR_006281
Software, algorithmCistromeLiu et al., 2011http://cistrome.dfci.harvard.edu/ap/; RRID:SCR_000242
Software, algorithmBEDToolsQuinlan and Hall, 2010http://bedtools.readthedocs.io/; RRID:SCR_006646
Software, algorithmJ-circosAn et al., 2015https://omictools.com/j-circos-tool; RRID:SCR_011798
Software, algorithmVENNYOliveros, 2007http://bioinfogp.cnb.csic.es/tools/venny/index.html
Software, algorithmRR Core Team, 2013https://www.r-project.org; RRID:SCR_001905
Software, algorithmDAVID Bioinformatics
Resources
Huang et al., 2009bhttps://david.ncifcrf.gov/; RRID:SCR_001881
Software, algorithmIGVRobinson et al., 2011http://software.broadinstitute.org/software/igv/; RRID:SCR_011793
Software, algorithmSTARDobin et al., 2013https://github.com/alexdobin/STAR; RRID:SCR_005622
Software, algorithmHTSeqAnders et al., 2015http://www-huber.embl.de/HTSeq/; RRID:SCR_005514
Software, algorithmDESeqAnders and Huber, 2010http://www-huber.embl.de/users/anders/DESeq/; RRID:SCR_000154
Software, algorithmWGCNALangfelder and Horvath, 2008https://labs.genetics.ucla.edu/horvath/htdocs/CoexpressionNetwork/Rpackages/WGCNA/; RRID:SCR_003302
Software, algorithmXCMSSmith et al., 2006http://www.bioconductor.org/packages/release/bioc/html/xcms.html; RRID:SCR_015538
Software, algorithmMetaboAnalyst 3.0Xia et al., 2015http://www.metaboanalyst.ca/; RRID:SCR_015539
Software, algorithmCAMERAKuhl et al. (2012)https://bioconductor.org/packages/release/bioc/html/CAMERA.html; RRID:SCR_011924
Software, algorithmPD1.4Thermohttps://www.thermofisher.com/order/catalog/product/IQLAAEGABSFAKJMAUH
Software, algorithmPathways to PCDL
(version B.07.00)
Agilent Technologies
Software, algorithmPCDL Manager
(version B.07.00)
Agilent Technologies
Software, algorithmProfinder
(version B.08.00)
Agilent Technologies
Software, algorithmPrismGraphPadv6; RRID:SCR_002798

Additional files

Supplementary file 1

Summary of H3K27me3 peak regions.

Summary of ChIP-seq experiments. Two criteria for peak calling: IP/input ≥ 2, signals spanning ≥3 kb. In total, 222 peak regions of H3K27me3 were reproducibly identified by four biological replicates at 3d. For each peak regions, information pertaining to chromosomal location and genes therein contained were given. ChIP-seq was using muscle tissues of 3d=old animals. Genotype: 5905.

https://doi.org/10.7554/eLife.35368.020
Supplementary file 2

Summary of CRISPR/Cas9-led gene mutagenesis and lifespan.

For each gene, two sgRNAs with indicated sequences, detailed deletion sites confirmed by Sanger sequencing, and PCR validation were shown. To name new CRISPR mutant, a superscript amended to the gene contained a letter c denoting CRISPR/Cas9-directed mutagenesis followed by the size of genomic deletion. Mutants were backcrossed (at least five generations) into 5905 (Flybase ID FBst0005905, w1118) to assure that phenotypes were not associated with any variation in background. Lifespan curves, together with 50% (median lifespan) and 10% survival data were listed. (for lifespan assay: 25°C; n ≥ 140 per genotype for curve; log-rank test).

https://doi.org/10.7554/eLife.35368.021
Supplementary file 3

Summary of transcriptomic analysis of PRC2 longevity mutants.

RNA-seq summary. This table summarizes five categories of genes and their transcriptional changes in long-lived PRC2 mutants of indicated genotype: (1) commonly upregulated and (2) downregulated genes in all PRC2 long-lived mutants; genes known to promote longevity upon upregulation, including (3) insulin pathway genes, (4) TOR signaling pathway genes, and (5) genes of miscellaneous pathways. RNA-seq was from muscle and head tissues of 30d-old male flies.

https://doi.org/10.7554/eLife.35368.022
Supplementary file 4

Summary of transcriptomic analysis of genes in glycolysis, citric acid cycle, pentose phosphorylation pathway, and oxidative phosphorylation.

This table summarizes metabolic genes and their transcriptional changes in long-lived PRC2 mutants of indicated genotype. RNA-seq was from head and muscle tissues of 3d- and 30d-old male flies.

https://doi.org/10.7554/eLife.35368.023
Supplementary file 5

Supplemental Materials and methods: Primer sequences used for the study.

https://doi.org/10.7554/eLife.35368.024
Transparent reporting form
https://doi.org/10.7554/eLife.35368.025

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